The overall goals are to elucidate the structure and mechanism of action of the heat-stable enterotoxin (ST) produced by Escherichia coli and possibly other enteric bacteria (Klebsiella, Yersinia, Proteus and Enterobacter) and to develop appropriate in vitro assay systems for ST that can be used to quantitate bacterial synthesis and excretion of substances with ST-like properties. The following in vitro assays are either in use in our laboratory now (1-3) or will be developed (if possible): (1) electrical potential change across isolated intestinal epithelium; (2) cyclic GMP concentration in intestinal epithelial cells; (3) guanylate cyclase activity in isolated intestinal epithelial cell membranes; (4) change in growth characteristics of intestinal crypt cells in culture and (5) displacement of radiolabelled toxin from specific membrane receptor sites. With the aid of these assay systems we will attempt to isolate the purified toxin, determine its chemical nature, sequence it (if a peptide) and identify its interactions with guanylate cyclase, cell membrane receptors and intestinal ion transport processes. In addition we will characterize and, when appropriate, purify products of ST minus mutants that retain biological activity in any one of the in vitro assay systems.